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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with acceptable restrictions Deviation from the guideline: - according to the guideline, animals of the satellite groups will not be mated and, consequently, are not used for the assessment of reproduction/developmental toxicity. In this tudy the animals of the recovery groups were mated. - it was not clear if a detailed clinical observation was carried out - female weights on day of delivery and day 4 post-partum were missing - no full gross pathology and histopathology were carried out. Histopathology of testes and epidydimis was not conducted - behaviour of pups was not recorded
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Remarks:
in accordance with GLP principles
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: nanomaterial: Citrate-capped silver nanoparticles, average size: 7.9±0.95 nm (TEM) , source: Nanotech (Korea)
Details on test material:
- Name of test material (as cited in study report): citrate-capped silver nanoparticles (provided by ABC Nanotech (Daejeon, Korea))
- Particle size: estimated to be 7.9 ± 0.95 nm (according to the manufacturer’s information and based on TEM)(The particle size was confirmed in the prerforming laboratory and was found to be in the designated range.)
- Zeta potential of AgNPs in water (10 ppm): –17.55 ± 4.16 mV (calculated; average ± standard deviation)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio (Gyeonggi-do, Korea)
- Age at study initiation: 8 weeks old
- Housing: two rats per cage were housed in stainless-steel cages during the administration period, and one female was housed with one male during the mating period. During the gestation and lactation periods, mated females and foetuses were housed individually in polycarbonate cages.
- Diet (ad libitum)
- Water (ad libitum)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5–23.5 °C
- Relative humidity: 47.7–62.0%
- Air changes: 12 times each hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The suspension of silver nanoparticles for oral administration was prepared by diluting the stock silver nanoparticles (20% w/v) with distilled water and adjusting for the volume of 10 mL/kg bw of the rats. Formulations were freshly prepared daily prior to use. Suspensions were routinely stable in solution during the experiments. The size distribution of the silver nanoparticles in suspension was measured using a submicron particle size analyser (NICOMPTM, Santa Barbara, CA, USA) and energy-filtering (EF) TEM using a LIBRA120 apparatus (Carl Zeiss, Jena, Germany). To obtain EF-TEM images, AgNPs dispersed in ethanol were spread on the TEM grid.
The average size distribution was 8.8 ± 5.2 nm when measured immediately after preparation and 7.7 ± 4.8 nm 1 day after preparation. TEM images
also supported the size distribution.

A daily application volume (10 mL/kg) was adjusted according to the most recent body weight and the vehicle control rats were treated with an equivalent volume of distilled water.
Details on mating procedure:
- M/F ratio per cage: one female was housed with one male during the mating period
- Length of cohabitation: generally, the maximal mating period was 2 weeks
- Proof of pregnancy: each morning, the females were examined for the presence of sperm and vaginal plug. Day 0 of pregnancy was defined as the day when a vaginal plug or sperm was found.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Male rats: 14 days before mating, 14 days during the mating period and 14 days of post-mating until necropsy (daily).
Female rats (maximum of 52 days): 2 weeks before mating, during the mating and gestation period, and during 4 days of lactation.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 125 and 250 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Main groups
Vehicle control group: 10 males/10 females
62.5 mg/kg: 10 males/10 females
125 mg/kg: 10 males/10 females
250 mg/kg: 10 males/10 females

Recovery groups:
Vehicle control group: 5 males/5 females)
250 mg/kg: 5 males/5 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the high dose of 250 mg/kg for 52-day treatment was selected based on the preliminary dosage test of this study, in which salivation was shown in a few of pregnant rats during the treatment period of 7 days. No deaths occurred during the 7-day administration period.
- Post-exposure recovery period in satellite groups: after completion of the treatment period, animals were maintained untreated during recovery for 14 days in the case of males and 16 days in the case of females.
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations checked: clinical signs including mortality, motility, general appearance and autonomic activity
During the lactation period, nursing behaviours of dams was observed.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights of both sexes were measured once a week during pre-mating, mating and recovery periods. During the pregnancy and lactation periods, body weights were measured at day 0 (day of pregnancy), gestation day 3, 6, 9, 12, 15, 18 and 20 for pregnant rats.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was measured during the pre-mating, pregnancy, lactation and recovery periods. During the mating period, food consumption was not measured.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Ag ANALYSIS
Tissues including liver, kidney and lung were obtained from four female rats sacrificed after 4 days lactation. The concentration of Ag in these tissues was analysed using ICP-MS (Elan6100/Perkin Elmer, Manhattan, NY, USA).

OTHER:
- gestation length was observed and recorded.
Oestrous cyclicity (parental animals):
For the examination of oestrus cycle, a vaginal smear was taken daily from each female during the pre-mating period. Regularity and length of the cycle was examined.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight and epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- on the parturition date, live and dead pups, sex, and external anomalies of live pups were observed and recorded.
- sex rate and survival rate at day 4 postpartum and body weight of live pups on day 0 (day of delivery) and day 4 postpartum were recorded.
- during the lactation period, viability of pups was observed.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At scheduled termination, all live males and females were necropsied after repeated dosing for at least 28 or more days, and at day 5 of lactation, respectively. In recovery groups, males and females were terminated at 14 days after the first scheduled sacrifice of animals of the main groups. For all animals, gross necropsy consisted of a complete external and internal examination and identification of all abnormal findings with external surface and all orifices, cranial cavity, external surface of the brain and spinal cord, nasal cavity and paranasal sinus, thoracic-, abdominal- and pelvic cavities, and their viscera, cervical tissues and organs. Organs with gross lesions were preserved in 10% neutral buffered formalin.

Absolute organ weights were measured and their relative organ weights (organ-to-body weight ratios) were calculated from the terminal body weight for the following organs of parent and recovery animals when they were sacrificed: liver, spleen, heart, lung, brain, thymus, kidneys (left and right), adrenal glands (left and right), thyroid, pituitary gland, salivary gland, ovaries (left and right), testis (left and right), epididymis (left and right), seminal vesicles and uterus.
On completion of the gross pathology examination, histopathological examination of tissues was carried out for liver, kidney, adrenal gland, heart, lung, spleen, prostate gland, vagina, thymus, thyroid gland, stomach, urinary bladder and pancreas. Histopathological examination was performed on the aforementioned tissues from animals in the vehicle control and 250 mg/kg dose groups.
At necropsy, the numbers of corpora lutea and implantation sites were counted in all females.
Postmortem examinations (offspring):
For the necropsy of day 4 postpartum pups, five pups of five females in the vehicle control and 250 mg/kg/day groups were selected randomly at schedule termination. Liver, kidney, lung and brain were collected to obtain samples for electron microscopy.
Statistics:
Statistical analyses were performed by comparing the treatment groups with the vehicle control group using SPSS 10.1 Base Statistical Analysis System (SPSS Korea Data Solution, Seoul, Korea). The data were presented as mean ± SD. Variance in the numerical data was checked using Levene’s test. If the variance was homogeneous, the one-way analysis-of-variance test was conducted to determine which pairs of group comparison were significantly different. If this test showed significance between the groups, the data were analysed by the multiple-comparison procedure of the Dunnet’s post hoc test.
Reproductive indices:
Mating index, fertility index and pregnancy rate were recorded.
Delivery rate was recorded.
Pre- and post-implantation losses were calculated.
Offspring viability indices:
no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- deaths were not observed in any treated or control group.
- alopecia was observed in one male in the 125 mg/kg-treated group at 37 days after oral administration and two males in the 250 mg/kg-treated
group 38 days after treatment; the rats did not recover. Alopecia was observed in two pregnant rats in the vehicle-treated group, one in the 62.5 mg/kgtreated group, two in the 125 mg/kg-treated group and six in the 250 mg/kg-treated group. Recovery did not occur, except for two rats in the 250 mg/kg-treated group.
- transient salivation was observed immediately after administration of silver nanoparticles in a female in the high-dose group (250 mg/kg/day) on day 1 after gestation. Salivation was not observed in any other female rat during the entire experimental period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- no statistically significant changes in weight gain in any of the treatment groups during the pre-mating and mating periods in male rats, or in females during the pre-mating, gestation and lactation periods.
- compared to the entire experimental period, the weight gain during the first week of treatment was markedly more in male rats, while no difference
between control and treated groups was observed. This tendency was not observed in female rats.
- in both male and female rats, there was no significant difference in food consumption during the entire experimental period including pre-mating, gestation and post-parturition periods
- early during the study, food consumption was much more in male rats than in female rats, perhaps reflecting the difference in body sizes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- no statistically significant difference in oestrus cycle between the groups in female rats.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- no statistically significant differences in mating, fertility and pregnancy rate among the groups, except that one non-pregnant rat and one premature birth were observed in the vehicle group
- no statistically significant differences were observed in the gestation period, number of corpora lutea and implantation, delivery rate, percentage of live and dead pups to implantations, preimplantation loss and post-implantation loss

ORGAN WEIGHTS (PARENTAL ANIMALS)
- in the recovery groups, a statistically significant increase in absolute and relative weights of liver was observed in males, and an increase in absolute weights of kidneys and adrenal gland was observed in females.
- absolute and relative weights of spleen, testes, brain, pituitary gland, lung, heart, thymus, thyroid gland, salivary gland, seminal vesicles and epididymis in the treated groups showed no significant differences from the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- yellow discolouration of lung was observed in one male in the 250 mg/kg/day group and multifocal white discolouration of spleen and splenomegaly was observed in one female in the 125 mg/kg/day group.

HISTOPATHOLOGY (PARENTAL ANIMALS)(control and high-dose groups (250 mg/kg)
- hepatocyte vacuolisation was observed in three treated female rats and in one rat from the control group
- interstitial inflammatory cells were observed in the kidney of three female rats in the treated group and three male rats in the control group.
- adrenal gland: cortical vacuolisation was found in three male rats from both the recovery and treated groups in the 250 mg/kg-treated groups.
- cholesterol granuloma in the lung was observed in two male rats in the treated and recovery groups. Granuloma was observed in two female rats in the treated group and one female rat in the recovery group.
- extramedullary haematopoiesis of the spleen was observed in three male rats from both the treated and recovery groups, and in one rat in the vehicle group.
- in other organs including brain, spinal cord, seminal vesicle, testis and ovary, lesions related with the AgNPs were not observed.
- lung granulomatous lesions were observed in 2 of 10 rats. It could not be confirmed whether the granulomatous lesion was related with silver nanoparticles treatment because it was evident only in two rats. Measurement of Ag in the lung revealed the apparent abundant accumulation compared to organs like the liver and kidney. The data were not sufficient to conclusively associate Ag accumulation with granulomatous lesion in the lung. Further study is necessary.

OTHER FINDINGS (PARENTAL ANIMALS)
- tissue distribution of silver in pregnant rats: the average quantity of silver in the livers of treated rats (1117.55 ± 381.68 ng/g) was increased by 34-fold compared to the level in control livers (33.54 ± 15.25 ng/g). The silver levels in lung (treatment value: 4461.22 ± 2726.42 ng/g; control value: 20.91 ± 7.47 ng/g) and kidney (treatment value: 449.78 ± 151.29 ng/g; control value: 34.81 ± 13.97 ng/g) were also significantly increased. Especially, the level of silver in the lungs of treated rats (4461.22 ± 2726.42 ng/g) was markedly increased compared to the level in liver or kidney.

Effect levels (P0)

open allclose all
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified Generation not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
- no statistically significant differences were observed in the number of live and dead pups, sex ratio and survival rate.

BODY WEIGHT (OFFSPRING)
- no statistically significant differences were observed in the body weights of pups on postnatal days 0 and 4.

GROSS PATHOLOGY (OFFSPRING)
- no statistically significant differences were observed in the number of neonates with external anomalies.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
According to the authors, there was no statistically significant difference in mating, fertility and pregnancy rate among the groups. No statistically significant differences were observed in the following parameters examined: gestation period, number of corpora lutea and implantation, delivery rate, number of live and dead pups, percentage of live and dead pups to implantations, preimplantation loss, post-implantation loss, sex ratio, survival rate, number of neonates with external anomalies, and body weights of pups on postnatal days 0 and 4.